The labeling of proteins with fluorescent substituents, most commonly done using green fluorescent protein (GFP) and its derivatives is an invaluable tool for tagging and visualizing proteins in v/vo. The abilityto genetically encode protein labels as with GFP combined with the supedor characteristics of small molecule fluorophores will significantly improve the in vivo tagging and visualization of proteins. This proposal aims to exploitthe high-affinity interaction between Mtx and DHFR to label proteins in vivo by fusing the protein of interest to DHFR and then labeling the protein with small- molecule Mtx conjugates. In Preliminary Results, we present work recently submitted for publication demonstrating the feasibility of this approach, labeling both a plasma membrane and a nuclear protein fused to DHFR with Mtx-Texas Red in Chinese Hamster Ovary (CHO) cells. We then outline our plans to develop orthogonal Mtx-DHFR variants via protein engineering in Aim t. In Aim 2, the modular Mtx-DHFR labeling approach is applied to CALl to distinguish between the role of myosin-lib localizad to the cell posterior to that localized to the lamella in cell motility, an experiment difficultto achieve with current labeling methodologies. Finally, inAim 3 we demonstrate the use of Mtx-DHFR labels for multi- color tagging and FRET applications and characterize the efficacy of these labels in comparison to GFP. PERFORMANCESITE(S)(organizationc,ity,state) Department of Chemistry, Columbia University, New York, NY Department of Biology, Columbia University, New York, NY KEYPERSONNELS. eelnstrucF]onUss. econtinuationpagesasneededto providetherequiredinformationinthe formatshownbelow. StartwithPrincipaInl vestigatorL. ista11otherkeypersonneinl aJphabetlocradlerla,stnameflrst_ Name Organization Roleon Project Virginia W. Cornish Columbia University PI Michael Sheetz Columbia University Co-P1 Larry Miller Columbia University Post-Doctoral Associate DisclosurePermissionStatemenLADeltcablteoSBtR/STTROnly. Seeinstructions.[] Yes [] No PHS398(Rev.05/01) Page 2 FormPage2 Principal lnvestigator/Progrem Oirec_ (Last_ first, middle): Cornish, Virginia W, The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page. RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page .................................................................................................................................................. 1 Description,